Bistability of an In Vitro Synthetic Autoregulatory Switch

The construction of synthetic biochemical circuits is an essential step for developing quantitative understanding of information processing in natural organisms. Here, we report construction and analysis of an in vitro circuit with positive autoregulation that consists of just four synthetic DNA strands and three enzymes, bacteriophage T7 RNA polymerase, Escherichia coli ribonuclease (RNase) H, and RNase R. The modularity of the DNA switch template allowed a rational design of a synthetic DNA switch regulated by its RNA output acting as a transcription activator. We verified that the thermodynamic and kinetic constraints dictated by the sequence design criteria were enough to experimentally achieve the intended dynamics: a transcription activator configured to regulate its own production. Although only RNase H is necessary to achieve bistability of switch states, RNase R is necessary to maintain stable RNA signal levels and to control incomplete degradation products. A simple mathematical model was used to fit ensemble parameters for the training set of experimental results and was then directly applied to predict time-courses of switch dynamics and sensitivity to parameter variations with reasonable agreement. The positive autoregulation switches can be used to provide constant input signals and store outputs of biochemical networks and are potentially useful for chemical control applications

Activating membranes

We present a general dynamical theory of a membrane coupled to an actin cortex containing polymerizing filaments with active stresses and currents, and demonstrate that active membrane dynamics [Phys. Rev. Lett \textbf{84}, 3494 (2000)] and spontaneous shape oscillations emerge from this description. We also consider membrane instabilities and patterns induced by the presence of filaments with polar orientational correlations in the tangent plane of the membrane. The dynamical features we predict should be seen in a variety of cellular contexts involving the dynamics of the membrane-cytoskeleton composite and cytoskeletal extracts coupled to synthetic vesicles

Apigenin, by activating p53 and inhibiting STAT3, modulates the balance between pro-apoptotic and pro-survival pathways to induce PEL cell death

BACKGROUND: Apigenin is a flavonoid widely distributed in plant kingdom that exerts cytotoxic effects against a variety of solid and haematological cancers. In this study, we investigated the effect of apigenin against primary effusion lymphoma (PEL), a KSHV-associated B cell lymphoma characterized by a very aggressive behavior, displaying constitutive activation of STAT3 as well as of other oncogenic pathways and harboring wtp53. METHODS: Cell death was assessed by trypan blue exclusion assay, FACS analysis as well as by biochemical studies. The latter were also utilized to detect the occurrence of autophagy and the molecular mechanisms leading to the activation of both processes by apigenin. FACS analysis was used to measure the intracellular ROS utilizing DCFDA. RESULTS: We show that apigenin induced PEL cell death and autophagy along with reduction of intracellular ROS. Mechanistically, apigenin activated p53 that induced catalase, a ROS scavenger enzyme, and inhibited STAT3, the most important pro-survival pathway in PEL, as assessed by p53 silencing. On the other hand, STAT3 inhibition by apigenin resulted in p53 activation, since STAT3 negatively influences p53 activity, highlighting a regulatory loop between these two pathways that modulates PEL cell death/survival. CONCLUSION: The findings of this study demonstrate that apigenin may modulate pro-apoptotic and pro-survival pathways representing a valid therapeutic strategy against PEL

Granulocyte-activating mediators (GRAM)

In the present study we investigated the capability of human epidermal cells to generate granulocyte-activating mediators (GRAM). It could be shown that human epidermal cells as well as an epidermoid carcinoma cell line (A431) produce an epidermal cell-derived granulocyte-activating mediator (EC-GRAM) which stimulates human granulocytes to release significant levels of toxic oxygen radicals as measured by a lucigenin-dependent chemiluminescence (CL). For further characterization of EC-GRAM the A431 cell line was used. Supernatants of A431 cells usually contained maximal EC-GRAM levels within 24 h of incubation. Factor production was enhanced by bacterial lipopolysaccharide (LPS), but not by silica particles and PHA. Moreover, freeze-thaw lysates of A431 cells and extracts of heat-separated human epidermis contained significant levels of EC-GRAM. Preincubation of granulocytes with EC-GRAM resulted in an enhanced response to subsequent stimulation with the chemotactic peptide f-met-phe. In contrast EC-GRAM did not affect the response to PMA or zymosan particles. However, EC-GRAM treated granulocytes were unresponsive to restimulation with EC-GRAM. Upon high performance liquid chromatography (HPLC) gel filtration EC-GRAM eluted within two major peaks exhibiting a molecular weight of 17 kD and 44 kD. According to its biochemical and biological properties EC-GRAM can be separated from other cytokines such as ETAF/-interleukin 1, interleukin 2, interferons, granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor (TNF). However, an antibody to human GM-CSF neutralized about 75% of the activity. These results indicate that EC-GRAM activity stimulating the generation of reactive oxygen species by granulocytes is probably due to GM-CSF

Temporary Sculpture: Activating the Ordinary

Invite students to use everyday objects as art making materials. Through this lesson students will be challenged to consider how everyday objects can be re- imagined as art making materials. In this lesson students will research and discuss every day materials from the art room as materials for sculptures. Students will also consider how the ordinary spaces around them could be activated by the addition of a sculpture.https://scholarscompass.vcu.edu/cstae_resource_early/1000/thumbnail.jp

Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells

Interaction of the TNFR-receptor associated factor TRAF1 with I-kappa B kinase 2 (IKK2, IKK-beta, IKBKB) and TRAF2 indicating a dose dependent regulatory function of TRAF1 for NF-kappa B signaling

IKK2 is one of the most crucial signaling kinases for activation of the transcription factor NF-kappa B. Since many NF-kappa B activating pathways converge at the level of IKK2, we searched for interaction partners of this kinase using the C-terminal part (aa 466-756) as bait in a yeast two-hybrid system. We identified the N-terminal part (aa 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner, which was subsequently confirmed in mammalian cells by coimmunoprecipitation experiments. However, this interaction seemed weaker than the interaction between TRAF1 and TRAF2, an important activating adapter molecule of NF-kappa B signaling indicating that relative levels of IKK2, TRAF1 and TRAF2 might be important for the final biological readout. Reporter gene and kinase assays using ectopic expression of TRAF1 indicated that it can have both activating and inhibiting functions for IKK2 and NF-kappa B. Co-expression of fluorescently tagged TRAF1 and TRAF2 at different ratios implied that TRAF1 can affect clustering and presumably the activating function of TRAF2 in a dose dependent manner

Gene deficiency in activating Fcγ receptors influences the macrophage phenotypic balance and reduces atherosclerosis in mice

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis

E-cadherin can limit the transforming properties of activating β-catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. But only Colorectal Cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of pancreas) have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve a Wnt deregulation and acquire a crypt-progenitor-cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with significantly higher expression of the β-catenin binding partner E-cadherin. This increased expression is associated with a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction of E-cadherin synergised with an activating mutation of β-catenin so there was now a rapid CPC phenotype within the colon and SI. Thus there is a threshold of β-catenin that is required to drive transformation and E-cadherin can act as a buffer to prevent β-catenin accumulation